猪带绦虫六钩蚴45W-4BX的高效表达、纯化及活性分析

High Expression,Purification and Activity Analysis of Taenia solium Oncosphere 45W-4BX

截去猪带绦虫六钩蚴45W-4B基因的N端信号肽和C端17个疏水氨基酸序列形成45W-4BX。设计特异性表达引物,PCR扩增目的片段,经BamH和EcoR酶切后与表达载体pGEX-4T-1连接转化BL21感受态细胞。用酶切及PCR扩增鉴定阳性克隆,测序证明阅读框是否正确。用IPTG诱导表达,产物进行SDS-PAGE电泳,Western blot分析其活性。包涵体经纯化、透析复性后作为包被抗原检测囊虫猪和囊虫病人血清中的4B抗体。结果表明,截断的4BX(351bp)基因在大肠杆菌中获得高效表达,表达产物为相对分子质量40000的融合蛋白,并能被囊虫病人血清所识别。ELISA检测表明,囊虫病人血清中含有高滴度的4B抗体,所以45W-4BX的表达产物有可能作为候选抗原用于囊虫病的诊断和免疫预防。

A 45W-4B truncated fragment .designed as 45W 4BX,was formed by removal of both N＇-terminal signal and C＇-terminal sequences encoding 17 hydrophobic amino acids, cloned into BamH 1 and EcoR 1 pre-digested expression vector pGEX-4T-1 and transformed into fresh competent BL21 cells, The positive transformants determined by PCR and sequencing were induced with IPTG. The results of SDS-PAGE and Western blot showed that the truncated fragment was highly expressed as a 40000 fusion protein in the form of inclusive bodies and that refolded GST-45W-4BX was recognized by sera from cysticercosis patients. The results indicated high titcr of antibody against 4B was present in cysticercosis patients＇sera. These suggest that recombinant 45W-4BX would be useful in diagnosis and prevention of cysticereosis patients.